This invention relates to treatment of disease of viral origin in warm-blooded vertebrates. More particularly, this invention is directed to the use of secoiridoid compounds naturally occurring in plants of the family Oleaceae and derivatives thereof.
The olive tree and other members of the Family Oleaceae have been documented as a source of medicinal substances since biblical times. Needless to say, many researchers have studied the cocktail of phytogenic substances produced by the olive and other members of the Family Oleaceae. One compound that has received attention from the research community is the secoiridoid glucoside oleuropein, a compound of the formula 
wherein R is glycosyl and R1 is 2-(3,4-dihydroxyphenyl) ethyl. Related secoiridoids wherein R1 is H, CH3 or 2-(4-hydroxyphenyl ethyl) are also known to be endogenous to many plant species of the family Oleaceae, although in lesser concentrations and in fewer identified species than the ubiquitous oleuropein.
Animal studies have revealed that oleuropein itself or as a component of extracts of plant tissues containing that compound exhibit both hypoglycemic and cardiovascular effects. It is also known that oleuropein can be acid hydrolyzed to produce (xe2x88x92)-elenolic acid, a compound which has been reported to have antiviral properties in vitro, but little, if any, activity in vivo.
The present invention is based on the discovery that secoiridoid glucosides of the formula 
wherein R is glycosyl and R1 is hydrogen or an ester-forming group are metabolized in vivo to the dextrorotatory form of elenolic acid [(+)-elenolic acid], a compound of the formula 
wherein R1 is hydrogen, a compound which is believed to be more available in vivo than the corresponding diasteromer(xe2x88x92)-elenolic acid of the formula 
The enhanced in vivo efficacy of the oleuropein metabolite, (+)-elenolic acid relative to the corresponding levorotatory compound is thought to be due, at least in part, to its reduced affinity for serum proteins and thus its greater availability for uptake by virus infected tissues. Antivirally effective blood levels of (+)-elenolic acid can also be achieved by administration of (+)-elenolic acid and its esters of the formula 
wherein R1 is hydrogen or a pharmaceutically acceptable ester-forming group and salts thereof, which can be prepared from secoiridoid glucosides naturally occurring in plant material of the family Oleaceae via extraction and controlled enzyme (glucosidase) hydrolysis and/or deesterification/transesterification reactions.
Thus, it is one object of the present invention to provide a method of treatment of disease of viral origin in warm-blooded vertebrates by administering antiviral compositions containing secoiridoid glucosides native to the plant family Oleaceae and derivatives thereof.
Another object of the invention is to provide oral dosage forms of secoiridoid glucosides of Olea europaea and derivatives thereof.
In another more particular aspect of this invention plant material of the family Oleaceae and extracts thereof containing naturally occurring oleuropein glucosides and enzyme hydrolysates thereof are administered in treatment of diseases of viral origin in warm-blooded vertebrates suffering from such diseases.
One further object of this invention is a method for establishing antivirally effective blood levels of (+)-elenolic acid by the administration of oleuropein glucosides native to the family Oleaceae or their derivatives via transesterification, diesterification and/or glucolysis.
The present invention is directed to a method of treatment of disease of viral origin in warm-blooded vertebrates and to pharmaceutical formulations for use in such treatment methods. The method comprises the step of administering to a vertebrate suffering from a disease of viral origin an antivirally effective amount of an antiviral composition comprising a compound of the formula 
and a pharmaceutically acceptable carrier therefor. In the above formula, the group R is glucosyl and R1 is hydrogen or a pharmaceutically acceptable ester-forming group. When R1 is hydrogen, the acid compound represented can be utilized in the form of one of its pharmaceutically acceptable salts.
There are many diseases of viral etiology that afflict man and animal. In man, diseases such as hepatitis, mononucleosis, shingles, herpes, influenza, the common cold and even certain types of leukemia are known to be of viral etiology. Viral infections are also common in many animal species, both in meat producing and in companion animals. Rotovirus infections plague swine. Cattle develop bovine rhinovirus infections (shipping fever) when subjected to conditions of stress. Canine parvovirus and feline leukemia virus are common viral infections in those companion animals species. Such diseases of viral origin can be treated with resultant reduction in clinical symptomology by therapeutic administration of antiviral compositions in accordance with this invention.
The antiviral compositions administered in accordance with this invention comprise a compound of Formula I or II above in combination with a pharmaceutically acceptable carrier. The compounds of Formula I wherein R1 is 2-(4-hydroxyphenyl)ethyl or 2-(3,4-dihydroxyphenyl)ethyl are naturally occurring compounds in many plants of the Family Oleaceae, including members of the genus Fraxinus, Syringa and the genus Ligustrum. Preferred plant sources of the naturally occurring secoiridoids of Formula I wherein R1 is 2-(4-hydroxyphenyl)ethyl and 2-(3,4-dihydroxyphenyl)ethyl are varieties of Olea europaea (the olive). Preferred varieties of Olea europaea as a source of secoiridoid glycosides for use in accordance with this invention are the varieties Manzanillo and Mission.
The most prevalent of the secoiridoid compounds in such varieties is the compound of Formula I wherein R1 is 2-(3,4-dihydroxyphenyl)ethyl and R is glucosyl, a compound given the common name oleuropein. That compound can be readily isolated from plant material, preferably ground leaves of the olive by aqueous or aqueous-alcoholic extraction at room temperature or above, preferably at elevated temperature of about 40 to about 100xc2x0 C.
Oleuropein can then be purified, for example, by chromatographic separation procedures. That compound can then be used to formulate antiviral compositions in accordance with this invention or to prepare other antivirally effective compounds represented by Formulas I or II. Thus, for example, oleuropein can be subjected, to base catalyzed transesterification wherein the R1 group 2-(3,4-dihydroxyphenyl)ethyl is exchanged with another pharmaceutically acceptable ester-forming group. The term xe2x80x9cpharmaceutically acceptable ester-forming groupxe2x80x9d as used in defining the present invention, refers to those ester-forming groups which when cleaved via esterase reactions in vivo produce substantially non-toxic, physiologically compatible alcohols. Suitable pharmaceutically acceptable ester-forming groups include C1-C8 lower alkyl, and substituted C1-C8 alkyl, benzyl, substituted benzyl wherein the substituents are halo, C1-C4 alkoxy, C1-C4 acyloxy, and the like. The compound of Formula I wherein R1 is hydrogen can be produced by esterase-mediated deesterification of oleuropein, typically in an aqueous medium at a pH between about 6 and about 8.5.
The compounds of Formula II are prepared from the corresponding compounds of Formula I by treatment with glucosidase, preferably that from the olive at a pH of about 4 to about 5. The compound of Formula II wherein R1 is hydrogen is (+)-elenolic acid.
The compounds of Formula I or II wherein R1 is hydrogen represent carboxylic acids and such acids can be used in accordance with this invention in the acid form or in the form of their pharmaceutically acceptable salts formed with organic bases or inorganic bases, such as ammonium, alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, and potassium carbonate. Of the salt forms,.the potassium and sodium salts are particularly preferred.
The antiviral compositions of the present invention can be administered orally or parenterally in an antivirally effective amount to treat, i.e., reduce the symptoms, of diseases of viral origin. Oral dosage forms can be in a solid or liquid form and comprise an antivirally effective amount of a compound of Formula I or Formula II above and a pharmaceutically acceptable carrier. Such dosage forms can be formulated from pure compounds of Formula I or Formula II, or they can be formulated from ground plant materials of the family Oleaceae, preferably leaves of Olea europaea, or aqueous or aqueous alcoholic extracts thereof. Thus, for example, extraction of dried olive leaves with two volumes of a 12-15% ethanol/water solution for 10 days at room temperature provides an extract containing about 70 to about 250 mg of oleuropein per two ounces of the liquid extract. The extract itself can be administered orally as an antiviral composition in accordance with the method of treatment of the present invention, or aqueous or aqueous alcoholic (preferably methanol or ethanol) extracts can be spray-dried to provide a dry powder which can be formulated into oral dosage forms with other pharmaceutically acceptable carriers.
The solid oral dosage form compositions in accordance with this invention are prepared in a manner well known in the pharmaceutical art, and comprise at least one compound of Formula I or Formula II associated with at least one pharmaceutically acceptable carrier. In making such compositions, the compound of Formula I or Formula II, either in pure form or as a component of ground plant material or extracts thereof, are usually mixed, diluted or enclosed within a carrier. The carrier can be in a solid form, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Alternatively, the carrier can be in the form of a capsule or other container to facilitate oral administration. Thus the solid oral dosage forms for administration in accordance with the method of this invention can be in the form of tablets, pills, powders or soft or hard gelatin capsules. Alternatively, the antiviral compositions in accordance with this invention for oral administration can be in liquid form wherein the pharmaceutically acceptable carrier is water or an aqueous alcoholic medium. The compositions for administration in the present method can also be formulated with other common pharmaceutically acceptable excipients, including lactose, dextrose, sucrose, sorbitol, mannitol, starches, gums, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, methylcellulose, water, alcohol and the like. The formulations can additionally include lubricating agents such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxybenzoates, sweetening agents or flavoring agents. Further the compositions of the present invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
Orally administered compositions are preferably formulated in unit dosage form with each dosage normally containing from about 30 to about 500 mg of a compound of the Formula I or Formula II, more typically about 100 to about 500 mg of such active compounds. The term xe2x80x9cunit dosage formxe2x80x9d refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical carrier/excipient. The preferred dosage levels of the compounds of Formula I/Formula II for treatment of viral infections in accordance with this invention depend on the route of administration, the nature of the active compound or combination of active compounds, and patient condition. When administered orally for treatment of viral disease, the compounds of Formula I or Formula II are administered at a dose of about 0.1 to about 15 mg/kg, more preferably about 0.2 to about 10 mg/kg of patient/animal body weight. In treatment of viral infections oral dosage forms in accordance with this invention can be administered 1 to 4 times a day, again depending on patient condition and the nature of the disease being treated.
Similar considerations bear on the dosage range for parenteral administration of compounds of Formula I or Formula II in treatment of viral infections in accordance with this invention. Parenteral doses are, however, typically lower than those required for antiviral efficacy via the oral route of administration. Thus, antiviral treatment can be achieved by parenteral administration of about 0.05 to about 3 mg/kg of patient/animal body weight. Parenteral formulations for use in accordance with the present invention are prepared using standard techniques in the art. They are commonly prepared as sterile injectable solutions, using a parenterally acceptable carrier such as isotonic saline solution or as a sterile packaged powder prepared for reconstitution with sterile buffer or isotonic saline prior to administration to a patient. The injectable formulation can contain from about 1 to 50 mg of a compound of Formula I or II per ml of formulation.
Administration of an antivirally effective amount of a composition comprising a compound of Formula I or Formula II and a pharmaceutically acceptable carrier in accordance with this invention produce antivirally effective blood levels of (+)-elenolic acid, a compound of the formula 
via a heretofore unappreciated in vivo hydrolysis, and in the case of the compounds of Formula I, stereoselective hydrolysis/rearrangement. Administration of antiviral compositions of a compound of Formula I as part of ground/dried native plant material, preferably olive leaves, or as an aqueous or alcoholic extract of olive leaves is a particularly preferred embodiment in accordance with this invention. While not wishing to be bound by theory, the level of antiviral activity associated with such antiviral compositions may derive from a synergistic antiviral effect with other natural components of the olive leaf, such as the flavonoids rutin, hesperidin, and luteolin-7-glucoside.